Somatostatin Ameliorates β-Amyloid-Induced Cytotoxicity via the Regulation of CRMP2 Phosphorylation and Calcium Homeostasis in SH-SY5Y Cells

Somatostatin is involved in the regulation of multiple signaling pathways and affords neuroprotection in response to neurotoxins. In the present study, we investigated the role of Somatostatin-14 (SST) in cell viability and the regulation of phosphorylation of Collapsin Response Mediator Protein 2 (CRMP2) (Ser522) via the blockade of Ca2+ accumulation, along with the inhibition of cyclin-dependent kinase 5 (CDK5) and Calpain activation in differentiated SH-SY5Y cells. Cell Viability and Caspase 3/7 assays suggest that the presence of SST ameliorates mitochondrial stability and cell survival pathways while augmenting pro-apoptotic pathways activated by Aβ. SST inhibits the phosphorylation of CRMP2 at Ser522 site, which is primarily activated by CDK5. Furthermore, SST effectively regulates Ca2+ influx in the presence of Aβ, directly affecting the activity of calpain in differentiated SH-SY5Y cells. We also demonstrated that SSTR2 mediates the protective effects of SST. In conclusion, our results highlight the regulatory role of SST in intracellular Ca2+ homeostasis. The neuroprotective role of SST via axonal regeneration and synaptic integrity is corroborated by regulating changes in CRMP2; however, SST-mediated changes in the blockade of Ca2+ influx, calpain expression, and toxicity did not correlate with CDK5 expression and p35/25 accumulation. To summarize, our findings suggest two independent mechanisms by which SST mediates neuroprotection and confirms the therapeutic implications of SST in AD as well as in other neurodegenerative diseases where the effective regulation of calcium homeostasis is required for a better prognosis.


Introduction
Alzheimer's disease (AD) is a progressive neurodegenerative disorder and the most common form of dementia in the elderly population. Standard clinical features of the disease include memory loss, abnormal social behavior, and deterioration of cognitive function [1][2][3]. AD is characterized by the formation of amyloid plaques, composed of abnormally truncated fragments of the amyloid precursor protein called β-amyloid (Aβ), and intracellular neurofibrillary tangles (NFT), consisting of hyperphosphorylated Tau protein [4,5]. The complex pathophysiology observed in AD is associated with the accumulation of plaques and the formation of NFTs, along with other pathological changes, resulting in synaptic dysfunction, excitotoxicity, dendritic spine loss and overall destabilization of the neural network [6,7]. The overaccumulation of Aβ is considered as the prominent cause of disease severity and neuronal cell death; however, the precise mechanism of interconnecting AD onset and progression is not fully understood, despite the identification of signaling pathways that exert determinant roles [8,9]. One such crucial signaling molecule that may represent a critical determinant is collapse response mediator hyper-influx of Ca 2+ , leading to the inhibition of calpain activity. Furthermore, SST inhibits the p35/p25-induced hyper-activation of CDK5 and the subsequent hyper-phosphorylation of CRMP2.

MTT Cell Viability Assay
To determine cell viability in response to Aβ, SH-SY5Y cells were processed for the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay, as previously described [42]. Briefly, differentiated SH-SY5Y cells were treated with increasing concentrations of Aβ 1-42 (0, 1, 5, 10 and 20 µM) or SST (0.4, 2 and 10 µM) alone, and with the combination of Aβ 1-42 (5 and 20 µM) and SST (10 µM) for 24 h. Post-treatment, the cells were washed with phosphate-buffered saline (PBS) and incubated for 2 h at 37 • C in the presence of 300 µg/mL of methyl-thiazolyl diphenyl-tetrazolium bromide solution (Sigma) prepared in serum-free DMEM. The cells were subsequently washed in PBS, and the resulting formazan formed in the cells was dissolved in 200 µL of isopropanol for 15 min on a rotating shaker. The changes in color were analyzed using a spectrophotometer at a wavelength of 570 nm, with the background absorbance measured at 650 nm. The results are presented as percentage changes between the treated versus the control group.

Caspase/Apoptosis Activity Assay
The Aβ 1-42 induced apoptosis in differentiated SH-SY5Y cells was analyzed using the Caspase-3/7 Green Apoptosis Assay kit (Essen Bioscience, Ann Arbor, MI, USA) following the manufacturer's instructions. Briefly, SH-SY5Y cells were treated with Aβ 1-42 (5 µM) alone or in combination with an increasing concentration of SST (0.4, 2, 10 µM) in the presence of a DNA intercalating dye NucView TM 488 (Essen Bioscience). The resulting fluorescence was analyzed in the IncuCyte TM live-cell imaging system (Essen Bioscience), and the Caspase-3/7 activity was assessed as an index of cells undergoing apoptosis using an IncuCyte basic analyzer (Essen Bioscience).

Live/Dead Cell Assay
The Aβ 1-42 -induced toxicity in the presence or absence of SST was also analyzed using a LIVE/DEAD Cell Vitality Assay (Thermo Fisher Scientific, Waltham, MA, USA), following the manufacturer's instructions. The differentiated SH-SY5Y cells were treated with Aβ 1-42 (5 µM) or SST (10 µM) alone or in combination for 24 h. Post-treatment, the cells were washed with PBS and collected in 0.05% trypsin-EDTA (Thermo Fisher Scientific). The cells were then re-suspended in 100 µL of PBS in the presence of C 12resazurin (20 ng/µL) and SYTOX dye (1 µM) and incubated for 15 min at 37 • C. Following incubation, the cells were immediately assessed on LSR II (BD Bioscience, San Jose, CA, USA) with excitation at 488 nm and emission at 530 and 570 nm, and analyzed using FlowJo workstation (BD Bioscience).

Immunofluorescence Immunocytochemistry
The control and treated cells were fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.1% Triton-X100 in PBS for 15 min at RT. Following three washes in PBS, the cells were blocked with 5% Normal Goat Serum (NGS) for 1 h at RT. The cells were then incubated with rabbit polyclonal primary antibody Ser522-CRMP2 (Cat# CP2191; ECM Bioscience) and mouse monoclonal βIII Tubulin (Cat# 801202; BioLegend) in 5% NGS overnight at 4 • C. Following the overnight incubation with the primary antibodies, the cells were washed with PBS and incubated with Alexa-conjugated secondary antibodies for 1 h at RT (1:200; Invitrogen). For nucleus visualization, the cells were incubated with Hoechst dye 33258 (0.5 µg/mL, Calbiochem, La Jolla, CA, USA) for 10 min at RT. The coverslips were then mounted onto the slides and photographed using a Zeiss LSM700 confocal microscope (Carl Zeiss, Oberkochen, Germany). Image panels were constructed using Carl Zeiss Zen software.

Agonist Treatment
SSTR2 and 4 specific non-peptide agonists (L-779976 and L-803087) were kindly provided by Dr S.P. Rohrer, Merck. Briefly, the differentiated SH-SY5Y cells were treated with SSTR specific agonists (3, 10, 30 nM) with or without Aβ for 24 h. Following treatment, the whole cell lysate prepared was processed to determine the expression levels and the activity of proteins of interest using Western blot analysis.

Fluo-4 Calcium Assay
The intracellular calcium levels were assessed using the Fluo-4 Direct TM calcium assay kit (Invitrogen) following the manufacturer's instructions. Briefly, the SH-SY5Y cells were plated onto a 96-well plate coated with Matrigel and differentiated with RA for up to 5 days. Following differentiation, the cells were incubated with an equal volume of 2 X Fluo-4 Direct TM calcium reagents (including probenecid) at 37 • C for 60 min. Following the loading of the dye, the cells were treated with Aβ 1-42 (5 or 20 µM) or SST (10 µM) alone and in a combination. The changes in the fluorescence intensity were measured (excitation at 494 nm and emission at 516 nm) in a spectrophotometer in a time-dependent manner for 50 cycles (20 s each). Untreated cells were used as internal control. The changes in absorbance are presented as a fold-difference between the treatment versus control (n = 3; each experiment represents an average of 3-6 independent readings).

Statistical Analysis
All results are presented as mean ± SD of a minimum of three independent experiments, as indicated. All statistical analyses have been performed in Graph Prism5.0. Student's t-test, or one-way analysis of variance (ANOVA) was used as indicated. * p < 0.05 against control or Aβ 1-42 treatment was taken into consideration as significant.

SST Inhibits Aβ 1-42 -Induced Toxicity in Differentiated SH-SY5Y Cells
To determine the cell viability of SH-SY5Y cells in response to Aβ 1-42 -induced toxicity, multiple approaches were applied. Initially, the overall cell metabolism was assessed using MTT assay as recently described [43]. As shown in Figure 1A, in response to increasing the concentration of Aβ 1-42 (1, 5, 10 and 20 µM), differentiated SH-SY5Y cells exhibited dose-dependent toxicity in comparison to controls. At lower doses, SST displayed no significant effect on cell viability, whereas, at the higher dose (10 µM), SST produced a cytotoxic effect post 24 hr treatment ( Figure 1B). However, differentiated cells treated with Aβ 1-42 (5 and 20 µM) in combination with SST (10 µM) display enhanced cell viability when compared to Aβ 1-42 alone ( Figure 1C). Next, we assessed the effect of Aβ 1-42 on cell viability by evaluating the activity level of caspase-3/7 as an index of apoptosis. As shown in Figure 2A, the SH-SY5Y cells treated with Aβ 1-42 displayed an increase in basal caspase-3/7 activity that was significantly different when compared to the control. In contrast, the cells treated with SST alone displayed inhibition of caspase-3/7 activity. As shown in Figure 2A, SST in combination with Aβ 1-42 displayed time-and concentration-dependent inhibition of caspase-3/7 activity when compared to the cells treated with Aβ 1-42 alone. These results suggest that SST mediates the inhibition of Aβ-induced apoptosis in differentiated SH-SY5Y cells. To determine the changes in metabolism as well as cell membrane integrity in response to the Aβ 1-42 -induced toxicity, a Live/Dead cell assay was performed in SH-SY5Y cells. Interestingly, the Live/Dead assay did not show significant changes in metabolic activity, which may be due to the metabolic demand of cells undergoing apoptosis ( Figure 2B,C). However, when assessed strictly for the cell membrane integrity, the Live/Dead cell assay showed an increasing trend in cell permeability upon treatment with Aβ 1-42 alone, albeit insignificantly, indicative of the toxic effect of Aβ ( Figure 2B).

Somatostatin Downregulates the Phosphorylation of CRMP2 at the Ser522 Site
Previous studies have demonstrated that SST, when used in combination with neuritepromoting drugs, including nerve growth factor (NGF), brain-derived nerve growth factor (BDNF), or RA, increases the neurite outgrowth and promotes the differentiation of various cells, including SH-SY5Y cells [40,44]. It is well known that CRMP2 plays a critical role in mediating tubulin stability and neurite outgrowth [45]. However, whether SST-mediated neurite growth and elongation is directly associated with the suppression of CRMP2 phosphorylation in Aβ 1-42 -induced toxicity model is not well understood. Accordingly, we sought to examine whether SST attenuates the Aβ 1-42 -induced hyperphosphorylation of CRMP2 using Western blot analysis. Differentiated SH-SY5Y cells were treated with increasing concentrations of SST (0.4, 2 and 10 µM) in the presence of Aβ 1-42 (5 µM). Vehicle treated cells or the cells treated with scrambled Aβ 42-1 were considered as controls.
Furthermore, to determine changes in site-specific phosphorylation, three phosphorylation sites of CRMP2 that have been previously reported to be hyperphosphorylated in AD patients were selected (Thr514, Ser522 and Thr555) [46]. As shown in Figure 3, the phosphorylation levels of Thr514-or Thr555-CRMP2 did not show a dose-dependent response to any of the concentrations of SST in combination with Aβ 1-42 (Figure 3, panels A, B, and D). Although the level of CRMP2 phosphorylation at site Ser522 was not relatively altered by Aβ 1-42 alone, it was significantly downregulated in the presence of SST in a dose-dependent manner, with a maximal reduction in the presence of SST at 10 µM (Figure 3, panels A and C). Therefore, based on the cell viability assay and site-specific Thr522-CRMP2 phosphorylation, all subsequent experiments were performed using 10 µM of SST. Note that the Ser522-phosphorylation level is inhibited in a dose-dependent manner with increasing concentration of SST in the presence of Aβ. The data represent the mean ± SD of three independent experiments. * p < 0.05 against Aβ 1-42 treated alone.

Somatostatin Inhibits the Activation of CRMP2 in the Presence of Aβ
To determine whether increased CRMP2 phosphorylation at Ser522 is associated with neurite formation, the subcellular distribution and colocalization of phosphorylated CRMP2 at Ser522 and neuronal tubulin marker βIII-tubulin was determined. In differentiated cells treated with scramble Aβ 42-1 , CRMP2-like immunoreactivity was confined primarily to the cell body, along with some punctuated staining in neurites ( Figure 4A). The cells were mostly devoid of any colocalization and displayed no detectable changes in the presence of SST. Conversely, treatment with Aβ 1-42 induced CRMP2 phosphorylation in neurites and showed colocalization with βIII-tubulin. However, following treatment with Aβ 1-42 in combination with SST, CRMP2-like immunoreactivity was decreased, while the cells exhibited an increase in the expression of βIII-tubulin. To further validate whether CRMP2 phosphorylation at Ser522 in the presence of Aβ is abolished by SST, differentiated SH-SY5Y cells were treated with SST alone or in combination with Aβ 42-1 or Aβ 1-42 , and the cell lysate prepared was processed for immunoblot analysis. As shown in Figure 4B, cells treated with SST displayed significant inhibition on Aβ 1-42 -mediated CRMP2 phosphorylation at Ser522 in comparison to cells treated with Aβ 42-1 . A quantitative analysis of the changes in CRMP2 phosphorylation (Ser522) was determined by a densitometric analysis ( Figure 4B). These results suggest that SST suppresses the subcellular distribution of CRMP2 in SH-SY5Y cells and prompt the dissociation from βIII-tubulin in neurite formation.

SST Inhibits the Aβ 1-42 -Induced Over-Expression of SSTR4
The biological effects of SST are mediated by binding to five different receptor subtypes (SSTR1-5). We recently reported the role of SSTR2 and 4 in promoting the RA-induced neuronal differentiation of SH-SY5Y cells [40]. Here, accordingly, we monitored the changes in the expression of SSTR2 and 4 following treatment with either Aβ 1-42 alone or in combination with SST. Scrambled Aβ 42-1 was used as a control. SH-SY5Y cells were treated with Aβ 42-1 (5 µM) and Aβ 1-42 (5 µM) in the presence and absence of SST (10 µM) for 24 h. Post-treatment, cell lysates were collected and processed for immunoblot analyses for the expression of SSTR2 and 4. As shown in Figure 5, the cells treated with Aβ 42-1 in the presence of SST exhibited an increase in SSTR2 expression without any discernible changes in SSTR4 expression. In contrast, the SH-SY5Y cells treated with Aβ 1-42 displayed an increased expression of both SSTR2 and 4 when compared to the cells treated with Aβ 42-1 .
In the cells treated with SST in combination with Aβ 1-42 , SSTR2 expression remained higher than Aβ 42-1 -treated cells but was comparable to the cells treated with Aβ 1-42 alone. Interestingly, the cells treated with Aβ 1-42 in combination with SST showed a significant reduction of SSTR4 expression when compared to cells treated with Aβ 1-42 alone. These results indicate SST-induced changes in subtype-specific receptor internalization, desensitization, and degradation.

SSTR-Subtypes-Mediated Changes in CRMP2 Phosphorylation
To determine which receptor subtype is involved in the SST-mediated inhibition of CRMP2 activation, differentiated SH-SY5Y cells were treated with SSTR2 and 4 specific agonists alone or in the presence of Aβ for 24 hr. Post-treatment, cell lysates collected from controls and treated cells were processed for Western blot analysis to assess CRMP2 phosphorylation. As shown in Figure 6A, in comparison to the control, CRMP2 phosphorylation increased significantly in cells treated with Aβ 1-42 . Receptor agonists induced concentration-dependent changes on CRMP2 phosphorylation in a receptor-specific manner. As shown in Figure 6A, in the absence of Aβ 1-42 , at the lowest concentration (3 nM), SSTR2-specific agonist (L-779976) inhibits CRMP2 phosphorylation at Ser522, whereas at higher concentrations (10, 30 nM), a moderate increase in CRMP2 phosphorylation was observed. The differentiated SH-SY5Y cells treated with SSTR2 agonist (3 nM) in the presence of Aβ 1-42 displayed inhibition of CRMP2 phosphorylation when compared to the cells treated with Aβ 1-42 alone. However, in the presence of Aβ 1-42 and SSTR2 agonist at higher concentrations (10 and 30 nM), no significant change in CRMP2 phosphorylation was observed when compared to Aβ 1-42 treatment alone. Notably, a higher concentration of SSTR2 agonist displayed no apparent difference in the levels of CRMP2 phosphorylation with or without Aβ 1-42 . As shown in Figure 6A, differentiated SH-SY5Y cells treated with SSTR4 agonist (L-803087) displayed significantly higher CRMP2 phosphorylation in comparison to controls. However, such enhanced status of CRMP2 phosphorylation was relatively higher at a lower concentration (3 nM), in contrast to a higher concentration, without any distinguishable difference between 10 and 30 nM. Next, we determined whether Aβ 1-42 activated CRMP2 phosphorylation is suppressed in the presence of SSTR4 agonist. As shown in Figure 6B, the status of CRMP2 phosphorylation in cells treated with SSTR4 agonist in combination with Aβ 1-42 exhibited a concentration-dependent increase that was significantly higher than both controls and cells treated with Aβ 1-42 alone.

Somatostatin-Mediated Inhibition of Ser522-CRMP2 Is Regulated Through the Calcium Pathway
Increased intracellular Ca 2+ accumulation is a well-documented mechanism of Aβmediated toxicity via inducing calpain activity, over-activation of CDK5, and hyperphosphorylation of CRMP2 at Ser522, leading to the disassembly of the CRMP2 complex. Previous studies have suggested that SST inhibits Ca 2+ by binding to SSTR2 [47][48][49]. To assess whether SST inhibits Aβ induced an increase in the Ca 2+ influx, and the intracellular Ca 2+ content was monitored using Fluo-4 in RA differentiated SH-SY5Y cells. In the cells treated with SST alone (10 µM), the intracellular Ca 2+ level was comparable to the control. The cells treated with Aβ 1-42 alone (5 µM) had no significant effect on intracellular Ca 2+ levels at early time points (data not shown), whereas treatment with Aβ 1-42 alone at a higher concentration of 20 µM induced a time-dependent increase in intracellular Ca 2+ level within a short treatment duration ( Figure 7A). The intracellular Ca 2+ influx was suppressed and maintained at a lower level in cells treated with Aβ 1-42 (20 µM) in combination with SST (10 µM) when compared to Aβ 1-42 alone ( Figure 7A). These results indicate that SST potentially inhibits an Aβ 1-42 -induced increase in the Ca 2+ influx and supports possible mechanisms of SST-mediated neuroprotection in Aβ-induced toxicity. Whether SST-mediated changes in the intracellular Ca 2+ affected resulted in changes in calpain expression and CDK5 activity and their downstream p35/25 expression is not known. As shown in Figure 7B-E, differentiated SH-SY5Y cells treated with Aβ 42-1 in combination with SST showed a significant increase in calpain expression in comparison to Aβ 42-1 alone. The calpain expression in differentiated SH-SY5Y cells upon treatment with Aβ 1-42 alone was not changed as compared to scramble. However, cells treated with Aβ 1-42 in combination with SST displayed a significant inhibition of calpain expression in comparison to the cells treated with Aβ 1-42 alone ( Figure 7C). The CDK5 expression was also increased in the presence of SST and Aβ 42-1 in combination when compared to the cells treated with Aβ 42-1 ( Figure 7D). In particular, the cells treated with Aβ 1-42 and SST together also resulted in a significant increase in CDK5 expression compared to the cells treated with Aβ 1-42 alone. Interestingly, such changes in calpain expression did not translate into changes in p35 expression. Instead, p35 expression increased significantly in the presence of SST in combination with Aβ 42-1 as well as with Aβ 1-42 alone or in combination with SST ( Figure 7E). Taken together, in differentiated SH-SY5Y cells, these events are supposed to be interconnected but function independently.

Discussion
We recently described the role of SST in RA-induced neurite growth in SH-SY5Y cells and established a possible interaction with the changes in MAP2/Tau and TUJ1, as well as an ERK1/2 signaling pathway. We also uncovered that the cells displaying colocalization between SST and TUJ1 exhibited a more extended neurite growth than cells devoid of colocalization [40]. The intact neurite formation is essential for a normal neuronal function. In contrast, disrupted neurite organizations are often observed in neurological diseases, including AD, and are associated with impaired cognitive function and memory loss. Whether SST is involved in improving neurite outgrowth and maintaining neuronal integrity in Aβ-induced neurotoxicity is not known. In the present study, using differentiated SH-SY5Y cells, we describe the role of SST in Aβ-induced toxicity and the molecular determinants, including CRMP2, Ca 2+ influx, CDK5, calpain, and P35/25, that might be associated with neurite outgrowth and cell viability. We demonstrate that SST improves cell viability and inhibits Aβ activated caspase 3/7 activity. We did not observe significant changes in metabolic activity as a proxy for Aβ-induced toxicity, and this might require higher concentrations of Aβ [50,51]. Furthermore, SST downregulates the influx of calcium level, which plays a pivotal role in the CDK5 activity. Our data suggest that SST mediates changes in CRMP2 phosphorylation and Aβ 1-42 -induced toxicity via the regulation of calcium in differentiated SH-SY5Y cells. This newly discovered mechanism might be involved in improving microtubules' organization and neurite outgrowth in AD pathogenesis.
Amongst the neuropeptides studied to date, SST is one of the most significant peptides that changes during the onset and progression of AD, with a consistent reduction in both the cerebrospinal fluid and brain tissues of AD patients [52][53][54][55][56][57][58][59]. We have previously reported the neuroprotective role of SST against various neurotoxic insults, such as pro-inflammatory lipopolysaccharide and Aβ 1-42 in a human cerebral micro-vessel cell line (hCMEC/D3), cultured cortical neurons, and cultured striatal neurons, as well as QUINand NMDA-induced excitotoxicity and cell death [43,[60][61][62][63]. An intracerebroventricular (i.c.v) infusion of Aβ in rats led to the significant reduction of SST-positive neurons in various brain regions, including the hippocampus and the temporal and frontoparietal cortex [64][65][66][67]. Furthermore, studies have also shown colocalization between the somatostatinergic-neurons and Aβ plaques in brain regions, including the amygdala, cortex, and hippocampus, of AD patients [68,69]. Saito et al. reported that the activity of a potent inhibitor of Aβ accumulation, neprilysin, was elevated following the introduction of SST, resulting in a subsequent reduction of Aβ aggregation [70]. Consistent with these observations, in the present study, the SST-induced amelioration of the toxic effect of Aβ was corroborated via various toxicity assays, including MTT, caspase-3/7 activity assay, and LIVE/DEAD toxicity assay. Collectively, these findings suggest a significant neuroprotective role of SST against Aβ-induced toxicity.
The impaired CRMP2 expression or activity may lead to a significant disruption in the overall neurite structure and a decline in cognitive function. CRMP2 is associated with various characteristics of neurite homeostasis, such as formation, outgrowth, and guidance, as well as maintaining the proper microtubule assembly by binding to the microtubule heterodimers and inducing polymerization while directly regulating tubulin GTPase activity [13,21,[71][72][73]. The hyperphosphorylation of CRMP2 has been observed in NFTs as well as in the soluble fragments of the brain tissues derived from AD patients [2,74]. Furthermore, transgenic mouse models of AD, including (PSEN1 (M146V) KI, Thy1.2-AβPP (swe) and triple (PSEN1 (M146V) KI, Thy1.2-AβPP (swe), and Thy1.2-tau (P301L), exhibit a significant increase in CRMP2 phosphorylation in the hippocampus and cortex [2]. On the other hand, other transgenic mouse models of AD, such as Tg2576, P301L, or P301s tau, fail to show an increase in CRMP2 phosphorylation, suggesting that the combination of AβPP and PSEN1 mutation may be a prerequisite for dysfunctional CRMP2 phosphorylation. Consistent with these studies and in support of SST-mediated neuroprotective and neurite outgrowth promoting effects, we observed here that SST downregulated CRMP2 hyperphosphorylation in the presence of Aβ 1-42 . Reduced CRMP2 phosphorylation, along with the increased expression of βIII-tubulin and its dissociation from CRMP2 in neurites upon treatment with SST, is an indication that tubulin is a prerequisite in the neurite elongation. It was not surprising to note that no significant elevation in CRMP2-Ser522 phosphorylation levels in SH-SY5Y cells was observed following treatment with Aβ 1-42 in our study. A previous study has reported that the phosphorylation of CRMP2 at the T555 site was significantly elevated in the presence of Aβ 1-40 in SH-SY5Y cells. The study reported no such changes at Thr514 and Ser522 sites and linked such variations to the Aβ species-dependent mechanism [75]. However, despite the differences in the CRMP2 phosphorylation levels, both Aβ 1-40 and Aβ 1-42 potentially impacted neurite length and elicit similar cellular outcomes [75]. Therefore, the role of Aβ 1-42 , Aβ 1-40 , or Aβ 25-35 on the phosphorylation of various CRMP2 sites could be potentially explored in future studies.
Increased activation of CRMP2 in the presence of SSTR2 and 4 specific agonists is surprising and warrants future research. We have previously shown that both SSTR2 and 4 internalize in response to ligand binding [76,77]. Our past studies have shown that SSTR2 exists predominantly as homodimers on the cell surface, whereas SSTR4 exists as both monomers and homodimers [76,77]. The inhibition of CRMP2 phosphorylation at this lower dose of SSTR2 agonist suggests that SSTR2 internalization is not prompted at this concentration, but triggered at a higher concentration. Moreover, in the presence of SSTR4 agonist, the receptor internalization is expected at all the concentrations used and followed by degradation, which may account for CRMP2 phosphorylation, which may be even higher in the presence of Aβ. We have previously demonstrated that SSTR2 and SSTR4 exist as homo-and heterodimers on the cell surface, whereas agonist treatment leads to changes in the receptor dimerization and enhanced internalization [76,77]. Consistent with these observations, it is highly possible that the dissociation of SSTR2 and 4 homo-and heteromeric complexes at the cell surface in response to receptor activation resulted in enhanced CRMP2 phosphorylation.
Previous studies have shown increased phosphorylation of CRMP2 by CDK5 and GSK3β in AD patients when compared to the age-matched controls [2]. CDK5 is a serine/threonine kinase that is activated upon association with its substrate p35 or p39. The abnormal CDK5 expression or activity has been closely associated with neurotoxicity in various neurodegenerative diseases, including AD, HIV neurotoxicity, and prion-related encephalopathies [37,78,79]. Furthermore, the disruptions in intracellular calcium homeostasis have also been associated with the onset and progression of AD and other amyloidogenic diseases, such as Parkinson's disease [80][81][82]. Various mechanisms have been suggested for the Aβ-mediated increase in calcium influx, including the disruption of lipid integrity [83], the formation of cation-selective channels by Aβ [81,84], or the activation of selective cell surface receptors to calcium [80,85,86]. These studies further emphasize that the Aβ-induced increase in calcium influx is not solely dependent on one particular pathway, but mediated through a complex network. In particular, the excess Ca 2+ influx in the presence of Aβ leads to the calpain-mediated truncation of CDK5 substrate p35 into the much more stable form of p25, leading to the prolonged activation of CDK5, followed by the hyper-phosphorylation of downstream mediators such as CRMP2 [35,87,88].
SST through SSTR2 is known to inhibit the Ca 2+ influx [47][48][49]. In agreement with previous studies, we found an increased expression of SSTR2 upon treatment with SST in the presence or absence of Aβ, supporting SSTR2 as the essential receptor involved in the SST-mediated inhibition of Ca 2+ influx. Although the observed inhibitory changes in Ca 2+ influx were not significantly different between Aβ 1-42 alone or in combination with SST-14 due to higher deviations, a similar trend was observed in all the experiments performed. We predict that the assay's high sensitivity and changes in the baseline due to experimental variability might be the reason for such observation. Furthermore, we have also observed a significant inhibition of calpain expression in cells co-treated with Aβ and SST compared to the cells treated with Aβ alone. This inhibition of calpain expression by SST did not result in the inhibition of CDK5 expression. Still, it resulted in a significant decrease of CRMP2 phosphorylation at Ser522, suggesting that SST might inhibit the hyperphosphorylation of CRMP2 by interfering with Ca 2+ homeostasis. Furthermore, as the CDK5-mediated phosphorylation of downstream targets such as CRMP2 depends on the activity rather than the expression level of CDK5, a significant change in CDK5 activity mediated by SST is conceivable, and future studies are warranted in this direction.
The activation of CRMP2 (Ser522) upon treatment with SSTR2 and 4 specific agonists, in contrast to SST-mediated attenuation, is intriguing. However, the molecular mechanism associated with such contradicting results is not known. Whether the SST-mediated suppression of CRMP2 (Ser522) phosphorylation is due to the direct or indirect activation of multiple SSTR subtypes warrants further research. Furthermore, it is possible that unlike SST, which is highly associated with the Ca 2+ uptake, specific SSTRs may work independently of the calcium pathway and via the modulation of downstream signaling pathways. Importantly, the role of other CRMP phosphorylation sites and isoforms, specifically CRMP5, cannot be avoided from the discussion. Previous studies have shown that CRMP5 inhibits neurite outgrowth and antagonizes CRMP2-mediated axonal and dendrite growth [89]. It is highly possible that SSTR2 and 4 agonists might inhibit CRMP5, resulting in enhanced CRMP2 phosphorylation.

Conclusions
In conclusion, the findings from the current study elucidate the mechanistic regulatory role of SST in intracellular calcium homeostasis, CRMP2 phosphorylation, and neurite formation and integrity. These observations corroborate the neuroprotective role of SST in neurotoxicity and neurodegenerative diseases by suggesting a novel mode of action. Furthermore, as disrupted calcium homeostasis is restricted to the neurodegenerative disease, the effective regulation of calcium levels by SST may have significant therapeutic applicability.